This proposal is based on our recent identification and cloning of cDNA's specific for the novel surfactant-associated protein Mr=6,000 (ASP-6-Phe). This protein, with a second hydrophobic surfactant peptide, SAP-6-Leu is associated with full biophysical activity of pulmonary surfactant lipids. We will now test the hypothesis that human SAP-6-Phe expression is temporally and hormonally regulated in the perinatal period and will test whether this occurs by transcriptional or translational mechanisms. The long-term goals of this proposal include the identification of the base sequences of the SAP-6-Phe gene which are involved in lung specific and developmental specific expression of this protein. The present proposal will complete the base sequence of the cDNAs and gene which encode human SAP-6-Phe. A 5' fragment of the cDNA clone has been used to isolate and partially characterize the base sequences of the gene for human SAP-6-Phe which will be analyzed at protein level using monoclonal antibodies which we have generated against SAP-6-Phe. Rabbit antisera generated against the synthetic peptides based on the NH2-terminal sequence have been prepared and will be utilized for SAP-6-Phe identification. We will determine cell specificity, temporal and hormonal control of expression of SAP-6-Phe in explant cultures of human fetal lung. The nature of the control of SAP-6-Phe expression will be determined using hybridization assay by both Northern blot and "slot blot" analysis to quantitate specific message. Transcriptional runoff assays will be utilized to determine whether increases in SAP-6-RNA are related to changes in transcriptional rates or message stability. The final goals of the project include the analysis of the 5' flanking regions of SAP-6-Phe gene to identify sequences involved in tissue- specific and hormone-dependent expression of SAP-6-Phe in developing lung. Transfection assays with chloramphenicol transferase (CAT) vector and transgenic mice will be utilized for deletion analysis of the SAP-6-Phe 5' sequences to determine their role in developmental and hormonal expression. These studies seek to clarify the structure of SAP-6-Phe gene(s) and nature of controls involved in ontogenic expression of mammalian surfactant specific proteins. Knowledge of the role of SAP-6-Phe in surfactant biology and control of SAP-6-Phe synthesis will be important in developing new strategies for therapy of hyaline membrane disease in newborn infants.